Bottom Line:
This is especially true in the area of nanomedicine, due to physicochemical properties, such as mechanical, chemical, magnetic, optical, and electrical properties, compared with bulk materials.The first goal of this study was to produce silver nanoparticles (AgNPs) using two different biological resources as reducing agents, Bacillus tequilensis and Calocybe indica.Cells pretreated with pifithrin-alpha were protected from p53-mediated AgNPs-induced toxicity.

Background: Recently, the use of nanotechnology has been expanding very rapidly in diverse areas of research, such as consumer products, energy, materials, and medicine. This is especially true in the area of nanomedicine, due to physicochemical properties, such as mechanical, chemical, magnetic, optical, and electrical properties, compared with bulk materials. The first goal of this study was to produce silver nanoparticles (AgNPs) using two different biological resources as reducing agents, Bacillus tequilensis and Calocybe indica. The second goal was to investigate the apoptotic potential of the as-prepared AgNPs in breast cancer cells. The final goal was to investigate the role of p53 in the cellular response elicited by AgNPs.

Methods: The synthesis and characterization of AgNPs were assessed by various analytical techniques, including ultraviolet-visible (UV-vis) spectroscopy, X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM). The apoptotic efficiency of AgNPs was confirmed using a series of assays, including cell viability, leakage of lactate dehydrogenase (LDH), production of reactive oxygen species (ROS), DNA fragmentation, mitochondrial membrane potential, and Western blot.

Results: The absorption spectrum of the yellow AgNPs showed the presence of nanoparticles. XRD and FTIR spectroscopy results confirmed the crystal structure and biomolecules involved in the synthesis of AgNPs. The AgNPs derived from bacteria and fungi showed distinguishable shapes, with an average size of 20 nm. Cell viability assays suggested a dose-dependent toxic effect of AgNPs, which was confirmed by leakage of LDH, activation of ROS, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in MDA-MB-231 breast cancer cells. Western blot analyses revealed that AgNPs induce cellular apoptosis via activation of p53, p-Erk1/2, and caspase-3 signaling, and downregulation of Bcl-2. Cells pretreated with pifithrin-alpha were protected from p53-mediated AgNPs-induced toxicity.

Conclusion: We have demonstrated a simple approach for the synthesis of AgNPs using the novel strains B. tequilensis and C. indica, as well as their mechanism of cell death in a p53-dependent manner in MDA-MB-231 human breast cancer cells. The present findings could provide insight for the future development of a suitable anticancer drug, which may lead to the development of novel nanotherapeutic molecules for the treatment of cancers.

f13-ijn-10-4203: PFT-α inhibits B-AgNPs- and F-AgNPs-induced ROS generation in a p53-independent manner.Notes: Cells were pretreated with PFT-α (10 μM) for 1 hour and then incubated with respective IC50 concentrations of B-AgNPs or F-AgNPs for 24 hours. The relative fluorescence of DCF was measured using a spectrofluorometer, with excitation at 480 nm and emission at 530 nm. All experiments were carried out in triplicate, and the experiments were repeated at least three times. Data are expressed as the mean relative gene expression ± SD of three independent determinations.Abbreviations: B-AgNPs, bacterium-derived AgNPs; Con, control; DCF, 2′,7′-dichlorofluorescein; F-AgNPs, fungus-derived AgNPs; IC50, half-maximal inhibitory concentration; PFT-α, pifithrin-alpha; ROS, reactive oxygen species; SD, standard deviation.

Mentions:
In previous experiments, exposure to both B-AgNPs and F-AgNPs led to phosphorylation of p53 and induction of p53-dependent apoptosis in MDA-MB-231 cells, through reduction of cell viability, enhanced LDH leakage, increased ROS generation, increased DNA fragmentation, and increased impairment of MTP (Δψm). These results confirm that p53 plays a major role in the activation of certain signaling transduction pathways that control the apoptotic pathway. In order to investigate whether the observed induction of p53 expression by B-AgNPs and F-AgNPs is responsible for these cellular responses, MDA-MB-231 cells were pretreated with PFT-α to block the activation of p53-mediated ROS production. To determine the effect of B-AgNPs and F-AgNPs on oxidative stress in the presence of PFT-α, we measured ROS generation using 2′,7′-dichlorofluorescein diacetate. B-AgNPs- and F-AgNPs-induced intracellular ROS generation was evaluated using intracellular peroxide-dependent oxidation of dichlorodihydrofluorescein diacetate to form fluorescent DCF. DCF fluorescence was measured in cells treated with B-AgNPs and F-AgNPs for 24 hours. Treatment of MDA-MB-231 cells with PFT-α resulted in a decline in both B-AgNP- and F-AgNP-induced ROS production (Figure 13). Herein, we used H2O2 as a positive control, in which MDA-MB-231 cells treated with H2O2 showed an increase in DCF fluorescence compared with the control. Conversely, in cells that were pretreated with PFT-α, H2O2-stimulated ROS generation was compromised. Taken together, these results suggest that p53 transcriptional activity is involved in ROS production upon treatment with AgNPs.

f13-ijn-10-4203: PFT-α inhibits B-AgNPs- and F-AgNPs-induced ROS generation in a p53-independent manner.Notes: Cells were pretreated with PFT-α (10 μM) for 1 hour and then incubated with respective IC50 concentrations of B-AgNPs or F-AgNPs for 24 hours. The relative fluorescence of DCF was measured using a spectrofluorometer, with excitation at 480 nm and emission at 530 nm. All experiments were carried out in triplicate, and the experiments were repeated at least three times. Data are expressed as the mean relative gene expression ± SD of three independent determinations.Abbreviations: B-AgNPs, bacterium-derived AgNPs; Con, control; DCF, 2′,7′-dichlorofluorescein; F-AgNPs, fungus-derived AgNPs; IC50, half-maximal inhibitory concentration; PFT-α, pifithrin-alpha; ROS, reactive oxygen species; SD, standard deviation.

Mentions:
In previous experiments, exposure to both B-AgNPs and F-AgNPs led to phosphorylation of p53 and induction of p53-dependent apoptosis in MDA-MB-231 cells, through reduction of cell viability, enhanced LDH leakage, increased ROS generation, increased DNA fragmentation, and increased impairment of MTP (Δψm). These results confirm that p53 plays a major role in the activation of certain signaling transduction pathways that control the apoptotic pathway. In order to investigate whether the observed induction of p53 expression by B-AgNPs and F-AgNPs is responsible for these cellular responses, MDA-MB-231 cells were pretreated with PFT-α to block the activation of p53-mediated ROS production. To determine the effect of B-AgNPs and F-AgNPs on oxidative stress in the presence of PFT-α, we measured ROS generation using 2′,7′-dichlorofluorescein diacetate. B-AgNPs- and F-AgNPs-induced intracellular ROS generation was evaluated using intracellular peroxide-dependent oxidation of dichlorodihydrofluorescein diacetate to form fluorescent DCF. DCF fluorescence was measured in cells treated with B-AgNPs and F-AgNPs for 24 hours. Treatment of MDA-MB-231 cells with PFT-α resulted in a decline in both B-AgNP- and F-AgNP-induced ROS production (Figure 13). Herein, we used H2O2 as a positive control, in which MDA-MB-231 cells treated with H2O2 showed an increase in DCF fluorescence compared with the control. Conversely, in cells that were pretreated with PFT-α, H2O2-stimulated ROS generation was compromised. Taken together, these results suggest that p53 transcriptional activity is involved in ROS production upon treatment with AgNPs.

Bottom Line:
This is especially true in the area of nanomedicine, due to physicochemical properties, such as mechanical, chemical, magnetic, optical, and electrical properties, compared with bulk materials.The first goal of this study was to produce silver nanoparticles (AgNPs) using two different biological resources as reducing agents, Bacillus tequilensis and Calocybe indica.Cells pretreated with pifithrin-alpha were protected from p53-mediated AgNPs-induced toxicity.

Background: Recently, the use of nanotechnology has been expanding very rapidly in diverse areas of research, such as consumer products, energy, materials, and medicine. This is especially true in the area of nanomedicine, due to physicochemical properties, such as mechanical, chemical, magnetic, optical, and electrical properties, compared with bulk materials. The first goal of this study was to produce silver nanoparticles (AgNPs) using two different biological resources as reducing agents, Bacillus tequilensis and Calocybe indica. The second goal was to investigate the apoptotic potential of the as-prepared AgNPs in breast cancer cells. The final goal was to investigate the role of p53 in the cellular response elicited by AgNPs.

Methods: The synthesis and characterization of AgNPs were assessed by various analytical techniques, including ultraviolet-visible (UV-vis) spectroscopy, X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM). The apoptotic efficiency of AgNPs was confirmed using a series of assays, including cell viability, leakage of lactate dehydrogenase (LDH), production of reactive oxygen species (ROS), DNA fragmentation, mitochondrial membrane potential, and Western blot.

Results: The absorption spectrum of the yellow AgNPs showed the presence of nanoparticles. XRD and FTIR spectroscopy results confirmed the crystal structure and biomolecules involved in the synthesis of AgNPs. The AgNPs derived from bacteria and fungi showed distinguishable shapes, with an average size of 20 nm. Cell viability assays suggested a dose-dependent toxic effect of AgNPs, which was confirmed by leakage of LDH, activation of ROS, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in MDA-MB-231 breast cancer cells. Western blot analyses revealed that AgNPs induce cellular apoptosis via activation of p53, p-Erk1/2, and caspase-3 signaling, and downregulation of Bcl-2. Cells pretreated with pifithrin-alpha were protected from p53-mediated AgNPs-induced toxicity.

Conclusion: We have demonstrated a simple approach for the synthesis of AgNPs using the novel strains B. tequilensis and C. indica, as well as their mechanism of cell death in a p53-dependent manner in MDA-MB-231 human breast cancer cells. The present findings could provide insight for the future development of a suitable anticancer drug, which may lead to the development of novel nanotherapeutic molecules for the treatment of cancers.